Gel electrophoresis is commonly used to separate by molecular size biological molecules, such as deoxyribonucleic acid ("DNA"), ribonucleic acid ("RNA") and proteins. To perform gel electrophoresis, a polymeric gel, such as polyacrylamide, is formed in a glass tube, or between spaced glass or plastic plates. The tube or plates are then placed in a chamber along with anode and cathode elements at the top and bottom of the gel. Sample wells formed in the top of the gel are first filled with running buffer solutions, and then with molecule samples prepared in a sample buffer that may contain a tracking dye. Electrophoretic running buffer solutions containing conductive ions are added to the chamber to make electrical contact between the gel, the samples in the wells and the anode and cathode elements. A voltage is then applied across the gel, which causes the sample molecules and any tracking dye to migrate toward the bottom of the gel, and separate into bands whose migration distance depends on molecular size.
The macromolecule migration rate through the gel generally depends upon five principle factors: (1) the gel porosity; (2) the applied electric field strength; (3) the electrophoresis temperature; (4) the macromolecule charge density; and (5) the macromolecule size and shape. For reproducible high resolution electrophoresis, these five factors generally must be precisely controlled from gel-to-gel and from sample-to-sample.
The first four factors generally do not pose a significant problem for separating nucleic acids. Manufactured precast electrophoresis gels may be used to maintain highly uniform gel-to-gel porosity, and numerous gel types are available for separating different macromolecules. In addition, modern electrophoresis equipment accurately controls temperature and voltage during separation. Further, DNA and RNA charge densities are very uniform because of the repeating structure of the anionic phosphate backbone. This charge uniformity leads to a precise inverse correlation of mobility with molecular length or base number, allowing nucleic acids varying by one base unit to be resolved by electrophoresis.
DNA base sequencing is one of the most useful embodiments of denaturing gel electrophoresis separations. In DNA base sequencing, the DNA sequencing product is denatured and the resulting single stranded DNA sample is applied to the electrophoresis gel. The native structural forms of DNA and RNA result from hydrogen-bonded interactions between complementary sequences of two strands of nucleic acid or between complementary regions in a single strand. These interactions must be completely disrupted prior to and during electrophoresis to eliminate secondary structure so that precise correlation of size with mobility is maintained.
Heat and organic solvents such as formamide and/or urea can be used in aqueous solutions to disrupt hydrogen bonds, resulting in denatured DNA and RNA. Thus, denatured DNA separations typically are performed in 6% polyacrylamide gels containing tris (hydroxy methyl) amino-methane, borate, ethylene diamine tetra-acetic acid ("TBE") gel and running buffer, pH 8.3 to 9.0, with 6 to 8 M urea and/or 2 to 12 M formamide acting as denaturants. In addition, DNA separations typically are performed at high operating temperature, typically 45 to 55.degree. C., to maintain fully denatured DNA.
DNA separations frequently are performed in gels 25 to 50 cm in length and about 0.4 mm thick, cast between two glass sheets. The gel is positioned vertically, and sample molecules, mixed after the sequencing reactions with a sample buffer concentrate, are applied into small sample wells near the top of the gel. These sample wells can be pre-formed in the gel or by use of a "sharks toothed comb," as described by Joseph Sambrook et al., Molecular Cloning: A Laboratory Manual 13.45-13.46 (2d ed. 1989).
The sample application area typically is filled with gel running buffer. The sample molecules, which are more dense than the running buffer, are carefully layered under the running buffer and on top of the exposed gel surface. To achieve sharp, well defined bands, the vertical depth of the sample should be minimized. Because DNA migrates faster in free solution in the loading buffer than it does in the gel, the DNA is concentrated and therefore sharpened at the gel surface before it penetrates into the gel. Nevertheless, dispersion and/or diffusion of the sample upward in the sample well will reduce the efficiency of this sharpening effect and limit the number of bases that can be clearly resolved on a given gel.
To increase resolution, electrophoresis may be performed using gels thinner than 0.4 mm, which create less band dispersion during electrophoretic migration. Recently, Novel Experimental Technology, Inc. ("NOVEX") developed the QuickPoint.TM. precast minigel (10 cm wide by 12.5 cm long and 0.25 mm thick) for DNA base sequencing. QuickPoint.TM. is prepared with 6% polyacrylamide, 7M urea and a neutral pH buffer that provides stable electrophoresis and storage conditions. QuickPoint.TM. gels have very high resolution capabilities, and can be operated with voltage gradients greater than 100 volts/cm, which allows from 60 to over 100 DNA bases to be resolved in less than 10 minutes within an 11 cm gel.
To realize the potential separation efficiency of such thin gels, the sample bands must be very sharp from the beginning of the electrophoresis run. Because the gel is very thin, however, it is more difficult to carefully layer the sample molecules on the gel and minimize dispersion during loading. Also, the sample molecules must be loaded quickly, because the first few samples begin diffusing into the buffer and the gel before the final samples have been loaded and before electrophoresis commences. It is therefore an object of the present invention to provide a method for applying denatured nucleic acid samples to the sample wells in a denaturing electrophoretic gel to maximize sample resolution and throughput.
Once electrophoresis begins and the molecules separate into bands, the bands may widen and begin to curve upward, further impairing fine resolution separation. Band sharpness and flatness are affected by, among other things, re-naturation and diffusion. In addition, any free ions in the sample increase the conductance of the sample region, causing a low voltage drop across the sample region, which increases separation times and further impairs flatness and sharpness.
Prior art electrophoresis systems use sample buffers to increase sample density and enhance band sharpness and flatness. High density samples quickly settle into the sample wells and speed sample loading, and consequently improve resolution. Ideally, an electrophoresis sample buffer provides several important functions:
1. Controls sample zone pH during electrophoresis; PA1 2. Controls ion and sample molecule movement during electrophoresis; PA1 3. Increases sample density and/or viscosity to aid sample loading into the sample wells; PA1 4. Provides tracking dye(s) to aid monitoring the progress of electrophoresis; PA1 5. Provides denaturing agent(s) to disrupt macromolecules to their primary structure; and PA1 6. Provides various chemical reducing and/or chelating agents to control sample chemistries.
Prior art sample buffers that provide all six functions are commonly used in discontinuous, reducing, sodium dodecyl sulfate polyacrylamide gel electrophoresis ("SDS-PAGE") developed by U. K. Laemmli, 227 Nature 680-86 (1970), and by NOVEX, NOVEX Catalogue 59-73 (1996). In particular, these prior art sample buffers concentrate the sample molecules into very sharp starting zones. This process, called "stacking," is controlled by the common buffering ion contained in the buffer system comprised of the gel, sample buffer and running buffers. The common buffering ion typically is an amine or substituted amine, such as tris (hydroxy methyl) amino-methane ("Tris") or bis-(2-hydroxyethyl) iminotris (hydroxymethyl) methane ("Bis-Tris"), respectively, with a pK.sub.a close to the desired pH of the buffer system for maximum pH control.
Stacking occurs when the anions used to titrate the Tris or Bis-Tris to the desired pH of the buffer system move faster than the sample molecules, and the anions in the running buffer are slower than the sample molecules. Under this condition, the sample molecules become concentrated, or stacked, and the extent of the stacking effect is proportional to the concentration of the leading anions in the gel and/or sample buffer. Stacking enhances the subsequent sharpness of the separations, and is critical to high resolution electrophoresis.
Prior art sample buffers for denaturing nucleic-acid electrophoresis, however, have not been designed to utilize this stacking effect. The primary reason is that the buffer systems used for nucleic acid electrophoresis generally are continuous, having the same buffering amines and titrating anions in the gel, running buffer and, occasionally, the sample buffer. For example, NOVEX's TBE-Urea sample buffer contains (1) TBE buffer; (2) urea, which acts as a denaturant; (3) ethylene diamine tetra-acetic acid ("EDTA"), which acts as chelating agent to bind divalent cations in the sample; and (4) Ficoll.TM. (type 400), a highly branched polysaccharide of 400 kDa that increases the sample density and viscosity and retards molecular diffusion.
Further, prior art sample buffers for denaturing nucleic acid polyacrylamide gel electrophoresis have been developed to enhance ion chelation, but not necessarily to improve stacking. For example, the Sequenase.TM. Version 2.0 DNA Sequencing Kit (United States Biochemical/Amersham Life Science) uses the most common prior art sample buffer (or stop solution) and method for denaturing DNA. These buffers contain (1) 95% formamide; (2) 20 mM EDTA, titrated with sodium hydroxide to pH 8.0; and (3) 0.05% bromophenol blue and 0.05% xylene cyanol FF. Formamide acts as a denaturant, and EDTA acts as an ion-chelating agent to bind magnesium ions, as required for sequencing enzyme activity and native DNA structure.
The Sequenase.TM. method for denaturing DNA samples combines 3.5 volumes of sample molecules with 4 volumes of the sample buffer/stop solution, resulting in 10.7 mM EDTA and 51% formamide in the sample wells. A common modification of the Sequenase.TM. method combines 6 volumes of sample molecules with 4 volumes of sample buffer/stop solution, resulting in 8 mM EDTA and 38% formamide in the sample wells.
In addition, Tabor et al. U.S. Pat. No. 4,795,699 ("Tabor") describes a DNA sequencing analysis in which a sample buffer containing 90% (volume/volume) formamide, 10 mM EDTA, and 0.10% (weight/volume) xylene cyanol is added to each sequencing reaction sample before gel electrophoresis. To prepare denatured DNA samples for sequencing analysis, Tabor's method combines 3 volumes of DNA sequencing reaction samples with 6 volumes of sample buffer, resulting in a 6.6 mM concentration of EDTA and 60% concentration of formamide in the sample wells of the sequencing gels.
Although the NOVEX TBE-urea sample buffer provides all but the stacking functions, and works reasonably well for standard denatured nucleic acid analyses, it does not provide maximum denaturing capacity when used in DNA sequencing. Additionally, TBE buffer has a relatively high pH (8.3) that hydrolyzes the urea amide groups, thereby creating highly conductive ions, changes in pH and reductions in denaturing strength that cause non-uniform results. Further, because all prior art sample buffers have pH ranges from 8 to 9, hydrolysis of urea and formamide is a problem for all prior art sample buffers.
Moreover, prior art sample buffers containing EDTA are titrated by adding sodium hydroxide. However, sodium hydroxide produces free sodium ions, which increases sample conductance, slows separation, increases heat generation, and enhances convective mixing and diffusion of sample molecules. It therefore also would be desirable to produce a neutral pH sample buffer that contains high EDTA concentration, but that has low sample conductance.
Further, the Sequenase.TM. and Tabor sample buffers enhance ion chelation, but do not sufficiently increase sample solution density. In addition, both buffers increase conductivity and therefore increase separation time. It therefore would be desirable to provide a sample buffer that enhances ion chelation, increases sample solution density, and decreases sample solution conductivity.
Additionally, although prior art sample buffers achieve some stacking and ion chelation, such systems have not been optimized for this purpose. It therefore also would be desirable to produce a sample buffer that enhances electrophoresis resolution by completely denaturing the sample molecule, enhancing ion chelation and stacking, and inhibiting diffusion.